Background: Beneficial effects of Yoga have been postulated to be due to modulation of the autonomic nervous system. Objective: To assess the effect of Isha Yoga practices on cardiovascular autonomic nervous system through short-term heart rate variability (HRV). Design of the Study: Short-term HRV of long-term regular healthy 14 (12 males and 2 females) Isha Yoga practitioners was compared with that of age- and gender-matched 14 (12 males and 2 females) non-Yoga practitioners. Methods and Materials: ECG Lead II and respiratory movements were recorded in both groups using Polyrite during supine rest for 5 min and controlled deep breathing for 1 minute. Frequency domain analysis [RR interval is the mean of distance between subsequent R wave peaks in ECG], low frequency (LF) power, high frequency (HF) power, LF normalized units (nu), HF nu, LF/HF ratio] and time domain analysis [Standard Deviation of normal to normal interval (SDNN), square of mean squared difference of successive normal to normal intervals (RMSSD), normal to normal intervals which are differing by 50 ms (NN50), and percentage of NN50 (pNN50)] of HRV variables were analyzed for supine rest. Time domain analysis was recorded for deep breathing. Results: Results showed statistically significant differences between Isha Yoga practitioners and controls in both frequency and time domain analyses of HRV indices, with no difference in resting heart rate between the groups. Conclusions: Practitioners of Isha Yoga showed well-balanced beneficial activity of vagal efferents, an overall increased HRV, and sympathovagal balance, compared to non-Yoga practitioners during supine rest and deep breathing.